This invention relates to a newly isolated mutant rpoH gene, to an altered .sigma..sup.32 protein encoded by the mutant rpoH gene, and to related E. coli strains. More particularly, the present invention relates to the use of such gene and related strains for enhanced accumulation of proteins synthesized by the gene.
The heat shock response occurs in wild-type Escherichia coli when cells are shifted abruptly from low temperature (e.g., 30.degree. C.) to high temperature (e.g., 42.degree. C.). Several of the heat shock proteins have been implicated in proteolysis of abnormal or heterologous proteins and thus are of applied interest. Most of the research with mutations in E. coli proteases and their effect on heterologous protein stability has involved the heat shock gene lon, encoding protease La. Protease La has been the most extensively studied protease in E. coli. In lon mutants, ATP-dependent proteolysis is reduced 2 to 4-fold. However, proteolysis is not prevented by lon mutants, even in strains which are lon null mutants, indicating that other bacterial proteases are also involved. The usefulness of lon mutants in accumulating higher levels of labile heterologous proteins appears to be marginal since, in most cases, the degradation of various heterologous proteins and peptides is not affected by lon null mutations. Exceptions, however, have been reported.
Transcription of all of the heat shock genes characterized to date is controlled by an alternate RNA polymerase sigma factor, the .sigma..sup.32 protein, the product of the rpoH (also known as htpR) gene. The sequence of the .sigma..sup.32 protein has been published, e.g., in Calendar et al., "Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma-32,"J. Bacteriology 170:3479-3484 (1988), which publication is incorporated herein by reference. The rpoH gene has been described, e.g., in Grossman, A. D. et al., "The hptR gene product of E. coli is a sigma factor for heat-shock promoters." Cell 38:383-390 (1984); Grossman, A. D. et al., "Analysis of Escherichia coli heat shock response," p. 327-331, in L. L. Lieve (ed.), Microbiology--1985, American Society for Microbiology, Washington, D.C. (1985); and Grossman, A. D. et al., ".sigma..sup.32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli", Genes Dev. 1:179-184 (1987); and reviewed by Helmann, J. D. et al. in "Structure and function of bacterial sigma factors." Annu. Rev. Biochem. 57:839-872 (1988); which publications are incorporated herein by reference. Escherichia coli cell growth at temperatures above 20.degree. C. requires the .sigma..sup.32 protein. Induction of heat shock gene expression is caused by a 20-fold increase in the amount of .sigma..sup.32 protein, resulting in exclusive transcription initiation from the heat shock gene promoters. Since the .sigma..sup.32 protein is relatively unstable, prolonged heat shock gene expression requires continual synthesis of .sigma..sup.32 protein. The heat shock proteins DnaK, DnaJ, and GrpE have been shown to negatively regulate heat shock gene expression by controlling the synthesis and stability of .sigma..sup.32. See Straus, D. B. et al., "DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of .sigma..sup.32." Genes Dev. 4:2202-2209 (1990).
Under permissive growth conditions (i.e., approximately 46.degree. C. or lower), the rpoH gene is transcribed from four tandem promoters, P1, P3, P4, and P5. Promoters P1, P4, and P5 are transcribed by RNA polymerase containing .sigma..sup.70 protein, the major sigma factor, and function at nonlethal temperatures. At lethal temperatures (e.g., 50.degree. C.), transcription of the rpoH gene occurs solely at P3 by RNA polymerase holoenzyme containing a novel sigma factor, .sigma..sup.E.
Mutations in the rpoH gene which curtail heat shock gene expression have been characterized. Such mutants might have practical value for synthesis of heterologous proteins because inhibition of the heat shock response causes an overall decrease in proteolysis. Besides protease La, other heat shock proteins have been implicated in proteolysis, including Clp, DnaK, DnaJ, GrpE, and GroEL. The precise functioning of DnaK, DnaJ, GrpE, and GroEL in proteolysis is still not well defined. As opposed to proteases La and Clp, it is doubtful that DnaK, DnaJ, GrpE, and GroEL are actual proteases. Rather, they appear to mediate proteolysis indirectly by yet undetermined mechanisms, perhaps involving folding/assembly. Heat shock proteins implicated in proteolysis (i.e., La, Clp, DnaK, DnaJ, GrpE, and GroEL) are synthesized at decreased rates in rpoH mutants. The most commonly used rpoH mutant allele, rpoH165, is an amber mutation that renders the cell temperature-sensitive for growth which is described in Cooper, S. et al., "A Temperature Sensitive Nonsense Mutation Affecting the Synthesis of a Major Protein of Escherichia coli K12", Molec. Gen. Genet. 139:167-176 (1975). Nearly all of the other rpoH mutant alleles characterized also provides a temperature-sensitive phenotype.
Mutations in the lon or rpoH gene phenotypically suppress a temperature-sensitive mutation in the .sigma..sup.70 protein subunit of RNA polymerase (rpoD800/rpoD285 mutant allele) at high temperature. The phenotypic suppression by lon or rpoH mutants at high temperature has been found to be due to decreased proteolysis of the mutant, labile .sigma..sup.70 protein. Of the four characterized rpoH mutants selected as temperature-resistant suppressor mutants of an rpoD800 strain, only one was found to be temperature-resistant in an rpoD.sup.+ background. As used herein, a superscript ".sup.+ " designates a wild-type gene. The rpoD gene is described in Helmann, J. D. et al., supra; and Yura, T. et al., "Genetic studies of RNA polymerase .sigma. factor in E. coli," p. 51-63, in Osawa, S. et al. (ed.) Genetics and Evolution of RNA Polymerase, tRNA, and Ribosomes. Univ. of Tokyo Press, Tokyo, Japan; which publications are incorporated herein by reference. The rpoD800 gene has been described by Hu, J. et al. in "Marker rescue with plasmids bearing deletions in rpoD identifies a dispensable part of E. coli .sigma. factor." Mol. Gen. Genet. 191:492-298 (1983), while mutations in the lon or rpoH gene which suppress the rpoD800 mutation have been described by Grossman, A. D. et al. in "Mutations in the lon gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase," Cell 32:151-159 (1983), and by Grossman, A. D. et al. in "Mutations in the rpoH (hptR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma-70 subunit of RNA polymerase," J. Bacteriology 161:939-943 (1985).
Thus, one drawback with using rpoH mutant strains is that the choice of mutant alleles is limited and a temperature shift to the non-permissive temperature, 42.degree. C., must be performed to maximize heterologous protein accumulation. Nonetheless, the use of rpoH mutant strains, particularly rpoH lon double mutants, has been reported to increase heterologous protein accumulation levels. Examples include IGF-1 and unstable derivatives of .lambda. repressor protein.
Conventionally, it is understood that altered rpoH genes that provide enhanced heterologous protein accumulation do so by decreasing proteolysis. Thus, for example, it has been reported recently in Goldberg, et al PCT/US89/03839, international application no. WO 90/03438, published Apr. 5, 1990, that an altered rpoH gene, rpoH165(am), has been discovered to result in improved heterologous gene expression by retarding proteolysis. Accordingly, researchers are still seeking to isolate and to characterize hitherto unknown rpoH mutations which result in higher accumulation levels of heterologous proteins. Also, it is important to identify an rpoH mutation which still confers desirable fermentation properties on the strain for optimal productivity of heterologous proteins, including 1) cell viability coupled with a relatively fast growth rate within a range of temperatures (e.g., 25.degree.-42.degree. C.), 2) lack of a mucoid phenotype characteristic of lon mutants, and 3) enhanced accumulation of various heterologous proteins without performing a temperature shift. The novel rpoH mutants described below possess all of these desired attributes.